A major disadvantage of drug administration by injection is that trained personnel are often required to administer the drug. For self-administered drugs, many patients are reluctant or unable to give themselves injections on a regular basis. Injection is also associated with increased risks of infection. Other disadvantages of drug injection include variability of delivery results between individuals, as well as unpredictable intensity and duration of drug action.
Despite these noted disadvantages, injection remains the only approved delivery mode for a large assemblage of important therapeutic compounds. These include conventional drugs, as well as a rapidly expanding list of peptide and protein biotherapeutics. Delivery of these compounds via alternate routes of administration, for example, oral, nasal and other mucosal routes, often yields variable results and adverse side effects, and fails to provide suitable bioavailability. For macromolecular species in particular, especially peptide and protein therapeutics, alternate routes of administration are limited by susceptibility to inactivation and poor absorption across mucosal barriers.
Mucosal administration of therapeutic compounds may offer certain advantages over injection and other modes of administration, for example in terms of convenience and speed of delivery, as well as by reducing or eliminating compliance problems and side effects that attend delivery by injection. However, mucosal delivery of biologically active agents is limited by mucosal barrier functions and other factors. For these reasons, mucosal drug administration typically requires larger amounts of drug than administration by injection. Other therapeutic compounds, including large molecule drugs, peptides and proteins, are often refractory to mucosal delivery.
The ability of drugs to permeate mucosal surfaces, unassisted by delivery-enhancing agents, appears to be related to a number of factors, including molecular size, lipid solubility, and ionization. Small molecules, less than about 300–1,000 daltons, are often capable of penetrating mucosal barriers, however, as molecular size increases, permeability decreases rapidly. Lipid-soluble compounds are generally more permeable through mucosal surfaces than are non-lipid-soluble molecules. Peptides and proteins are poorly lipid soluble, and hence exhibit poor absorption characteristics across mucosal surfaces.
In addition to their poor intrinsic permeability, large macromolecular drugs, including proteins and peptides, are often subject to limited diffusion, as well as lumenal and cellular enzymatic degradation and rapid clearance at mucosal sites. These mucosal sites generally serve as a first line of host defense against pathogens and other adverse environmental agents that come into contact with the mucosal surface. Mucosal tissues provide a substantial barrier to the free diffusion of macromolecules, while enzymatic activities present in mucosal secretions can severely limit the bioavailability of therapeutic agents, particularly peptides and proteins. At certain mucosal sites, such as the nasal mucosa, the typical residence time of proteins and other macromolecular species delivered is limited, e.g., to about 15–30 minutes or less, due to rapid mucociliary clearance.
Various methods and formulations have been attempted to enhance the absorption of drugs across mucosal surfaces. Penetration enhancing substances that facilitate the transport of solutes across biological membranes are widely reported in the art for facilitating mucosal drug delivery. Mucosal penetration enhancers represented in these reports include (a) chelators (e.g., EDTA, citric acid, salicylates), (b) surfactants (e.g., sodium dodecyl sulfate (SDS)), (c) non-surfactants (e.g., unsaturated cyclic ureas), (d) bile salts (e.g., sodium deoxycholate, sodium taurocholate), and (e) fatty acids (e.g., oleic acid, acylcarnitines, mono- and diglycerides). Numerous additional agents and mechanisms have been proposed for enhancing mucosal penetration of drugs. These include, for example, reducing the viscosity and/or elasticity of mucus layers that cover mucosal surfaces; facilitating transcellular transport by increasing the fluidity of the lipid bilayer of membranes; altering the physicochemical properties (e.g., lipophilicity, stability) of drugs; facilitating paracellular transport by altering tight junctions across the epithelial cell layer; overcoming enzymatic barriers; and increasing the thermodynamic activity of candidate drugs.
While many penetration enhancing methods and additives have been reported to be effective in improving mucosal drug delivery, few penetration enhanced products have been developed and approved for mucosal delivery of drugs. This failure can be attributed to a variety of factors, including poor safety profiles relating to mucosal irritation, and undesirable disruption of mucosal barrier functions.
In view of the foregoing, there remains a substantial unmet need in the art for new methods and tools to facilitate mucosal delivery of biotherapeutic compounds. Related to this need, there is a compelling need in the art for methods and formulations to facilitate mucosal delivery of biotherapeutic compounds that have heretofore proven refractory to delivery via this route, to avail the medical community of the numerous potential advantages of mucosal drug delivery.
One group of therapeutic compounds of interest for mucosal delivery is a therapeutic peptide designated peptide YY. Peptide YY (PYY) as used herein is a class of peptides, peptide analogs, peptide conjugates and peptide mimetics exemplified in base structure and activity by a prototypic, 36 amino acid peptide having tyrosine residues at both C- and N-terminals. The paired terminal tyrosine residues in this well-known prototypic peptide accounts for the “YY” designation used in the art. Structural analyses have demonstrated approximately 70% homology between PYY, neuropeptide Y, and pancreatic polypeptide, suggesting a common evolutionary precursor. These three peptides together form the primary members of the so-called Pancreatic Polypeptide (PP) family believed to play an important role in the normal physiology of the brain-gut axis. PP peptides all exhibit C-terminal amidation, a feature common in many biologically active peptides.
PYY co-localizes with glucagon and glucagon-like products within endocrine L cells of the intestinal mucosa and to a lesser extent in alpha cells of the pancreas. Studies in a number of mammals including humans have shown that PYY expression increases sequentially along the length of the intestines, with peptide levels in the rectum up to 100-fold greater than in the duodenum. This unique distribution makes PYY an ideal candidate for hormonal regulation of upper gastrointestinal function. In fact, PYY causes decreased gastric acid secretion, delays gastric emptying and slows intestinal transit time. PYY is also known to inhibit exocrine and possibly endocrine functions of the pancreas.
Release of PYY occurs following a meal. An alternate molecular form of PYY is PYY3-36. Eberlein, et al. Peptides 10: 797–803, 1989; Eysselein, et al., Peptides 11: 111–116, 1990; Grandt, et al. Regul Pept 51: 151–9, 1994, each incorporated herein by reference. This fragment constitutes approximately 40% of total PYY-like immunoreactivity in human and canine intestinal extracts and about 36% of total plasma PYY immunoreactivity in a fasting state to slightly over 50% following a meal. It is apparently a dipeptidyl peptidase-IV (DPP4) cleavage product of PYY. PYY3-36 is reportedly a selective ligand at the Y2 and Y5 receptors, which appear pharmacologically unique in preferring N-terminally truncated (i.e. C-terminal fragments of) NPY analogs. Peripheral administration of PYY reportedly reduces gastric acid secretion, gastric motility, exocrine pancreatic secretion, gallbladder contraction and intestinal motility. Yoshinaga, et al. Am J Physiol 263: G695–701, 1992; Guan, et al. Endocrinology 128: 911–6, 1991; Pappas, et al. Gastroenterology 91: 1386–9, 1986; Savage, et al. Gut 28: 166–70, 1987, each incorporated herein by reference. The effects of central injection of PYY on gastric emptying, gastric motility and gastric acid secretion, as seen after direct injection in or around the hindbrain/brainstem may differ from those effects observed after peripheral injection. Chen and Rogers. Am J Physiol 269: R787-R792, 1995; Chen, et al. Regul Pept 61: 95–98, 1996; Yang and Tache, Am J Physiol 268: G943–8, 1995; Chen, et al. Neurogastroenterol Motil 9: 109–116, 1997, each incorporated herein by reference. For example, centrally administered PYY had some effects opposite to those described herein for peripherally injected PYY3-36 in that gastric acid secretion was stimulated, not inhibited. Gastric motility was suppressed only in conjunction with TRH stimulation, but not when administered alone, and was indeed stimulatory at higher doses through presumed interaction with PP receptors. PYY has been shown to stimulate food and water intake after central administration. Morley, et al. Brain Res 341: 200–203, 1985; Corp, et al. Am J Physiol 259: R317–23, 1990; U.S. application 20020141985, each incorporated herein by reference.
One of the earliest reported central effects of neuropeptide Y (NPY) was to increase food intake, particularly in the hypothalamus. Stanley, et al. Peptides 6: 1205–11, 1985. PYY and PP are reported to mimic these effects, and PYY is more potent or as potent as NPY. Morley, et al., Brain Res 341: 200–203, 1985; Kanatani, et al. Endocrinology 141: 1011–6, 2000; Nakajima, et al. J Pharmacol Exp Ther 268: 1010–4, 1994, U.S. application 20020141985, each incorporated herein by reference.
Receptors for PYY (designated as Y1, Y2, Y5) have been identified throughout the gastrointestinal tract, including both small bowel and colon mucosal epithelium. These findings raise the possibility that PYY may also exhibit additional actions on gastrointestinal tissues, including regulation of cell growth. Dysregulation of cell growth is most critical in the development and progression of cancer. Prospective clinical applications of PYY exist for therapy of malignant disease and cancer. Tseng et al., Peptides 23: 389–395 (2002), Michel, et al. Pharmacol Rev 50:143–50, 1998; Gehlert, Proc Soc Exp Biol Med 218: 7–22, 1998, each incorporated herein by reference.
Additional prospective clinical applications of PYY or neuropeptide Y exist for therapy of controlled food intake or obesity. Food intake is regulated by the hypothalamus, including the melanocortin and neuropeptide Y (NPY) systems in the arcuate nucleus. The orexigenic NPY and the anorectic alpha melanocyte-stimulating hormone (α-MSH) systems of the hypothalamic arcuate nucleus are involved in the central regulation of appetite. However, the potential mechanisms that link signaling associated with meal ingestion with these hypothalamic-feeding circuits are unclear.
From the prototypic PYY peptide, a subpeptide PYY3-36 is formed as a cleavage product produced by the action of didpetidyl peptidase-IV. PYY3-36 is a major circulating species exhibiting a distinct pharmacology, and showing antidiabetic and antiobesity actions in several animal models. This gut-derived hormone is released postprandially in proportion to calories ingested. PYY3-36 shares 70% amino-acid sequence identity with NPY and acts through NPY receptors. The NPY Y2 receptor (Y2R), a putative inhibitory presynaptic receptor, is highly expressed on NPY neurons in the arcuate nucleus, which is accessible to peripheral hormones, although not expressed on the neighboring pro-opiomelanocortin (POMC) neurons. Peptide PYY3-36, a high affinity Y2R agonist, is released from the gastrointestinal tract postprandially in proportion to the calorie content of a meal.
Studies have investigated the effects of peripheral administration of PYY3-36 on feeding. Experiments show that peripheral injection of PYY3-36 in rats inhibits food intake and reduces weight gain. PYY3-36 also inhibits food intake in mice but not in Y2r-null mice, which suggests that the anorectic effect requires the Y2R. Peripheral administration of PYY3-36 increases c-Fos immunoreactivity in the arcuate nucleus and decreases hypothalamic Npy messenger RNA. Intra-arcuate injection of PYY3-36 inhibits food intake. PYY3-36 also inhibits electrical activity of NPY nerve terminals, thus activating adjacent pro-opiomelanocortin (POMC) neurons. In humans, infusion of normal postprandial concentrations of PYY3-36 significantly decreases appetite and reduces food intake by 33% over 24 h. Thus, postprandial elevation of PYY3-36 may act through the arcuate nucleus Y2R to inhibit feeding in a gut-hypothalamic pathway. Nature 418: 650–654, 2002, incorporated herein by reference.
Experiments have been performed to investigate whether peripheral PYY3-36 might inhibit food intake through the Y2R in the arcuate nucleus, an area that is directly accessible to circulating hormones. To investigate this hypothesis, experiments injected PYY3-36 directly into the arcuate nucleus. In rats fasted for 24 h, food intake was significantly decreased by doses as low as 100 fmol, which resulted in a similar inhibition to that seen after intraperitoneal administration. To establish whether these effects occurred through the Y2R, a Y2R selective agonist, Y2A (N-acetyl [Leu, Leu] NPY24-36) was used. Its affinity was confirmed using receptor-binding studies on cell lines that expressed the NPY Y1, Y2 and Y5 receptors. Intra-arcuate nucleus injection of Y2A in rats previously fasted for 24 h dose-dependently inhibited (100 fmol to 1 nmol) food intake. To confirm the anatomical specificity of this effect, Y2A (100 fmol to 1 nmol) was injected into the paraventricular nucleus (PVN) of rats fasted for 24 h and found no alteration of food intake. To define further the role of the Y2R in the feeding inhibition caused by peripheral PYY3-36, the effect of PYY3-36 on Y2r-null mice and littermate controls was examined. PYY3-36 inhibited daytime feeding in a dose-responsive manner in fasted male wild-type mice but did not inhibit food intake in fasted male Y2r-null mice. Nature 418: 650–654, 2002, incorporated herein by reference.
Results of this experiment suggest that the cells in the arcuate nucleus detect circulating peripheral satiety signals and relay these signals to other brain regions. This is supported by the observation that leptin modifies the activity of both the proopiomelanocortin (POMC) and NPY arcuate neurons. Experiments have shown, through a combination of electrophysiological and hypothalamic explant studies, that the gut hormone, PYY3-36, can directly influence hypothalamic circuits, which results in coordinate changes in POMC and NPY action. In addition, PYY3-36 administered directly into this brain region reduces food intake. Data show that postprandial concentrations of PYY3-36 inhibit food intake in both rodents and man for up to 12 h, which suggests that PYY3-36 has a role in longer term regulation of food intake. This contrasts with previously characterized gut-derived short-term satiety signals such as cholecystokinin, the effects of which are relatively short-lived. The failure of PYY3-36 to inhibit food intake in the Y2r-null mice provides further evidence that PYY3-36 reduces food intake through a Y2R-dependent mechanism. Experimental results suggest that a gut-hypothalamic pathway that involves postprandial PYY3-36 acting at the arcuate Y2R has a role in regulating feeding. Thus, the PYY3-36 system may provide a therapeutic target for the treatment of obesity. Nature 418: 650–654, 2002, incorporated herein by reference.
Leptin is an adiposity hormone that modulates the activity of multiple hypothalamic signaling pathways involved in the control of food intake. Experiments were designed to evaluate whether central administration of leptin or one of its downstream mediators, neuropeptide Y (NPY), could affect food intake by modulating the brain stem neurophysiological response to ascending meal-related feedback signals in the nucleus of the solitary tract (NTS) in anesthetized male Long-Evans rats. NTS neurons at the rostrocaudal level of the area postrema were dose-dependently activated by gastric loads ranging from 2–10 ml, and leptin and NPY had opposite modulatory effects on this load volume/activity relationship: leptin significantly increased NTS responses to gastric loads, whereas NPY reduced the potency and efficacy with which gastric loads activated NTS neurons. These effects were probably not mediated by peripheral effects of centrally administered peptides or by the gastrokinetic effects of central NPY or leptin, because the dose-response relationship between gastric load volume and neurophysiological firing rate was unchanged in gastric load-sensitive vagal afferent fibers. These data suggest a mechanistic framework for considering how feeding behavior occurring in meals is altered by challenges to energy homeostasis, such as fasting and overfeeding. Endocrinology 143: 3779–3784, 2002, incorporated herein by reference.
As noted above, the subpeptide PYY3-36 exhibits antidiabetic and antiobesity actions in several animal models. Since gastric emptying is an important mediator of post-prandial glycemia, experiments investigated whether PYY3-36 affected gastric emptying, and specifically investigated whether such effect was mediated via the area postrema (AP), a circumventricular organ with no blood-brain barrier, that is accessible to circulating peptides and known to be involved in the regulation of gastrointestinal function. While saline injected AP-lesioned animals had a tendency to delay gastric emptying compared to non-operated and sham operated rats, PYY3-36 administration had no additional effect on gastric emptying rate in the AP-lesioned animals. PYY3-36 has a potent effect to inhibit gastric emptying in normal rats via a pathway that appears to include the area postrema. American Diabetes Association, 62nd Annual Scientific Sessions, Jun. 14–18, 2002; Abstract 1661-P, incorporated herein by reference.
To investigate a possible role for peripherally administered PYY3-36 in metabolic control, experiments examined the effects of its infusion for 28 days via subcutaneous osmotic pumps (at 0, 30, 100, 300 and 1000 μg/kg/d) to C57BL/6 mice (n=14–22/group) previously fed a high fat diet (HF; 58% fat vs LF, low-fat control diet, with 11% fat) for 47 days. Contrasting with previously reported effects of centrally administered PYY and PYY3-36 to increase food intake and body weight, the results of this study indicate that peripherally administered PYY3-36 exhibits an anti-obesity and glucose-lowering effect in diet-induced obese mice. American Diabetes Association, 62nd Annual Scientific Sessions, Jun. 14–18, 2002; Abstract 1718-P, incorporated herein by reference.
Additional studies examined the effect of 4 weeks of continuous infusion of PYY3-36 on 8 week-old male Zucker Diabetic Fatty (ZDF) rats. The results suggest that the improvement in glycemic control following chronic PYY3-36 in ZDF rats was unlikely to be due to direct effects on glucoregulatory hormone secretion, and may instead be related to mechanisms such as reduced nutrient availability. The dissociation of food consumption from weight gain in these experiments, despite the presence of glucose-lowering effects, suggests factors such as caloric loss in the urine of diabetic animals. Further studies in Diabetic Fatty Zucker (ZDF) rats demonstrated that continuous infusion of PYY3-36 decreased food consumption and improved glycemic control, without decreasing body weight gain, but those observations did not account for caloric redistribution due to treatment-related changes in glycosuria. In contrast to previous data in diabetic animals where caloric losses in the urine may have been a factor and where there was no change in body weight, the results of the present study indicate that in the absence of glycosuria in obese non-diabetic fa/fa rats, PYY3-36 treatment decreases food consumption and body weight. American Diabetes Association, 62nd Annual Scientific Sessions, Jun. 14–18, 2002; Abstract 1717-P; Abstract 2499, incorporated herein by reference.
Further experiments examined the effect of continuous infusion of PYY3-36 in genetically obese mice (ob/ob; B6.V-Lep ob) on food intake and body. Chronic PYY3-36 infusion in ob/ob mice was associated with a dose dependent decrease in body weight compared to saline infused animals. This effect appeared to be independent of any effect on food intake. (American Diabetes Association, 62nd Annual Scientific Sessions, Jun. 14–18, 2002; Abstract 1716-P, incorporated herein by reference.
As part of an investigation of the potential involvement of the gut in these actions, other experiments examined the dose-response of subcutaneously injected PYY3-36 on pentagastrin-stimulated gastric acid secretion. The effect of PYY3-36 on ethanol-induced gastritis was evaluated in fasted male Sprague Dawley rats. To study acid secretion, rats chronically implanted with gastric cannulae were injected subcutaneously with 125 μg/kg pentagastrin. Gastric contents obtained by flushing the cannulae every 10 min were titrated to measure acid production. PYY3-36 dose-dependently and potently inhibited pentagastrin-stimulated gastric acid secretion, manifest also as a gastroprotective effect, in rats. Endogenously circulating PYY3-36 may play a physiologic role in controlling gastric acid secretion and protecting the gastric mucosa. (American Diabetes Association, 62nd Annual Scientific Sessions, Jun. 14–18, 2002; Abstract 2444-PO, incorporated herein by reference.
Obesity and its associated disorders are common and very serious public health problems in the United States and throughout the world. Upper body obesity is the strongest risk factor known for type 2 diabetes mellitus, and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, arteriosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, cancers of the breast, prostate, and colon, and increased incidence of complications of general anesthesia. Kopelman. Nature 404: 635–43, 2000, incorporated herein by reference. It reduces life-span and carries a serious risk of co-morbidities above, as well disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypertension hypercholesterolemia, cholelithiasis, orthopedic injury, and thromboembolic disease. Rissanen, et al. BMJ 301: 835–7, 1990, incorporated herein by reference. Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or “Syndrome X.” Recent estimates for the medical cost of obesity and associated disorders are $150 billion worldwide. The pathogenesis of obesity is believed to be multifactorial but the basic problem is that in obese subjects nutrient availability and energy expenditure do not come into balance until there is excess adipose tissue. Obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. A therapeutic drug useful in weight reduction of obese persons could have a profound beneficial effect on their health.
Previous methods have attempted to increase weight gain in healthy subjects by administering peptide YY to the subject. Additional reports propose treatment of metabolic disorders such as obesity, diabetes, and increased cardiovascular risk by administering peptide YY or a peptide YY agonist. See e.g., U.S. Pat. No. 5,912,227; U.S. application 20020141985. These and related reports have attempted to administer peptide YY by various routes. Notably, there are no previous descriptions that demonstrate a therapeutically effective nasal formulation or method for administering peptide YY for any of the foregoing diseases and conditions.
Previous attempts to successfully deliver peptide YY for therapeutic purposes have suffered from a number of important and confounding deficiencies. These deficiencies point to a long-standing unmet need in the art for pharmaceutical formulations and methods of administering peptide YY that are stable and well tolerated and that provide enhanced delivery to target sites, e.g., the serum or other selected cellular or extracellular compartment, in mammalian subjects.